1. Field of the Invention
The present invention relates generally to the field of cytokine actions and more particularly concerns methods and compositions for inhibiting and modulating the actions of CXC intercrine molecules. Disclosed are peptide compositions which inhibit interleukin 8 (IL-8) and, particularly, which preferentially inhibit IL-8-induced release of degradative enzymes by neutrophils. These compositions may be employed to treat various inflammatory diseases and disorders including the Adult Respiratory Distress Syndrome (ARDS) and cystic fibrosis.
2. Description of the Related Art
IL-8 is a member of the CXC intercrine family of cytokines, so named due to elements of their N-terminal sequences. This family also includes, amongst others, peptide molecules known as growth related oncogene (GRO, or GRO/MGSA) and macrophage inflammatory protein 2xcex2 (MIP2xcex2). IL-8 is a peptide of approximately 8 kD, and is about 72 amino acids in length, with this length varying according to the post-translational processing in different cell types (Yoshimura et al., 1989; Hebert et al., 1990; Strieter et al., 1989). The IL-8 gene was first identified by analyzing the genes transcribed by human blood mononuclear cells stimulated with Staphylococcal enterotoxin A (Schinid and Weissman, 1987). IL-8 production is known to be induced by tumor necrosis factor and interleukin 1 (Strieter et al., 1990).
MGSA/GROxcex1 is a peptide which was first identified as an autostimulatory growth factor of Hs 294T melanoma cells (Richmond et al., 1983; 1985; Richmond and Thomas, 1986). Further studies indicated that it was produced by diverse melanoma cell lines (Lawson et al., 1987) and played an important role in the tumorigenesis and growth of malignant melanoma cells (Mintz and Silvers, 1993). Sequence analysis demonstrated that MSGA/GROxcex1 is a member of the superfamily called xe2x80x9cxcex1-chemokinesxe2x80x9d (Oppenheim et al., 1991). This family of proteins were chemotactic for neutrophils and had substantial sequence homology, a C-X-C motif near the amino-terminal end, and two additional Cys residues closer to the carboxyl-terminal end. The functions of xcex1 chemokines were mediated by receptors on the cell surface membrane (Murphy and Tiffany, 1991; Holmes et al., 1991; Mueller et al., 1994). Recent studies showed the presence of some types of chemokine receptors on melanoma cells. Some melanoma cells possess interleukin-8 (IL-8) receptors similar to those on neutrophils (Holmes et al., 1991; Mueller et al., 1994; Moser et al., 1993; Norgauer et al., 1996).
MGSA/GROxcex1 is a 73 amino acid peptide which shares sequence characteristics of a superfamily of peptides called xcex1-chemokines. Richmond and colleagues initially discovered that melanoma cells secreted autostimulatory (autocrine) growth factors (Richmond et al., 1983; 1985; Richmond and Thomas, 1986; Richmond et al., 1982). It was found that most of the activity was caused by a single acid stable protein of about 15 kDa and designated it melanoma growth stimulatory activity (MGSA) (Richmond et al., 1983; 1985). MGSA was found to be a mitogen for the melanoma cell line Hs 294T which produces this factor. MGSA was secreted by diverse melanoma cell lines but not by benign nevus cell lines (Lawson et al., 1987), while immunoreactive MGSA was shown in both types of cells (Richmond et al., 1986). cDNA for MGSA isolated from Hs 294T cells was later found to be identical to oncogene growth-related peptide (GROxcex1) gene (Richmond et al., 1988). The formal name for this protein was then designated as MGSA/GROxcex1.
Recently, a second chemokine was found to be important for melanoma cell growth and metastasis in some melanoma cell lines. Schadendorf and colleagues determined that some melanoma cell lines tested secreted IL-8 (Schadendorf et al., 1993). Both of two IL-8 secreting cell lines studied in more detail were dependent on IL-8 for growth. Antisense oligonucleotides targeted against human IL-8 mRNA inhibited cell proliferation, colony formation in soft agar, and secretion of IL-8 into culture supernatants. In an analysis of 13 different human melanoma cell lines, it was shown that expression of IL-8 correlates with the metastatic potential of melanoma cells in BALB/c nude mice (Singh et al., 1994).
Other studies further indicate a role of these chemokines in melanoma growth and tumorigenesis. Mintz and Silvers developed a method of producing melanomas by grafting skin from Tyr-SV40E transgenic mice which are highly susceptible to melanoma to Tyr-SV40E hosts of a low susceptibility of the same inbred strain (Mintz and Silvers, 1993). It was suggested that growth factors and cytokines known to be produced in wound repair may trigger the growth and malignant conversion of melanocytes. Nanney and colleague showed that MGSA/GROxcex1 and its receptors are present in human burn wounds and may act as a mediator for wound repair (Nanney et al., 1995). MGSA/GROxcex1 and Ill-8 was induced by ultraviolet B radiation in human keratinocyte cell lines (Venner et al., 1995)
IL-8 interacts with at least two distinct receptors on neutrophils (Holmes et al., 1991; Murphy and Tiffany, 1991). The receptors are coupled to GTP-binding proteins, allowing transmission of the IL-8 signal into the cell (Wu et al., 1993). While most of the members of the intercrine family, such as GRO and MIP2xcex2, bind to one of the receptors, IL-8 binds to both of the IL-8 receptors (LaRosa et al., 1992; Cerretti et al., 1993). The three dimensional structure of IL-8 has been elucidated by NMR (Clore et al., 1990) and by X-ray crystallography (Clore and Gronenbom, 1992; Baldwin et al., 1991). A freely movable amino terminal end is followed by three beta pleated sheets and an alpha helix is located at the carboxyl-terminal end (Oppenheim et al., 1991). Several lines of evidence suggest that both the amino- and carboxyl-terminal ends are involved in binding to its receptors (Clore et al., 1990; Clark-Lewis et al., 1991; Moser et al., 1993).
Certain functions of the CXC intercrines have been elucidated by several laboratories (Yoshimura et al., 1989; Schroder et al., 1988; Peveri et al., 1988). For example, the major functions of the IL-8 peptide appear to be related to its ability to stimulate neutrophil chemotaxis and activation (Larsen et al., 1989; Schroder et al., 1988; Peveri et al., 1988; Yoshimura et al., 1987) and to promote angiogenesis (Koch et al., 1992). If neutrophils are xe2x80x98primedxe2x80x99, e.g., by agents such as surface adherence or E. coli endotoxin (also known as lipopolysaccharide or LPS), IL-8 also stimulates the release of neutrophil enzymes such as elastase and myeloperoxidase.
Although the neutrophil inflammatory response is essential for the destruction of bacteria which are invading the body, inappropriate neutrophil activation causes several problems. For example, if the neutrophils are properly primed when attracted to the lungs, they release destructive enzymes into the lung tissue. This can lead to the development of adult respiratory distress syndrome (ARDS) (Weiland et al., 1986; Idell et al., 1985). ARDS attacks between 150,000 and 200,000 Americans per year, with a mortality rate of 50-80% in the best clinical facilities (Balk and Bone, 1983). ARDS is initiated by bacterial infections, sudden severe dropping of the blood pressure (shock), and many other insults to the body. Recent studies have demonstrated that IL-8 is the major neutrophil activator in the lungs of patients with ARDS (Miller et al., 1992), and primate models of endotoxin shock also implicate IL-8 as a causative agent (Van Zee et al., 1991).
High concentrations of IL-8 have also been found in inflammatory exudates in other disorders and pathological conditions in which IL-8 is thought to play an important pathogenic role (Brennan et al., 1990; Miller and Idell, 1993; Miller et al., 1992). For example, IL-8 has also been implicated as a possible mediator of inflammation in rheumatoid arthritis (Brennan et al., 1990; Seitz et al., 1991) and pseudogout (Miller and Brelsford, 1993); and to have a role in cystic fibrosis (McElvaney et al., 1992; Nakamura et al., 1992; Bedard et al., 1993). Therefore, modulation of IL-8 function appears to be good strategy to control a variety of pathological conditions.
Some progress has recently been made in identifying compounds capable of reducing IL-8 synthesis. Such compounds include IL-4, oxygen radical scavengers, secretory leukoprotease inhibitor and interferon gamma (Standiford et al., 1990; DeForge et al., 1992; McElvaney et al., 1992; Cassatella et al., 1993a; 1993b), however, such studies do not concern IL-8 inhibitors. Other diverse compositions, including protein kinase C inhibitors, IL-4, and anti-IL-8 antibodies, have also been reported to modulate IL-8 actions (Lam et al., 1990; Standiford et al., 1992; Mulligan et al., 1993). Unfortunately, these compounds are far from ideal as candidates for use as IL-8 inhibitors in a clinical setting.
Certain progress has also been made in identifying peptide IL-8 inhibitors, however, most of such work has focused on portions of the IL-8 molecule itself (Miller et al., 1990; Gayle et al., 1993). For example, the present inventors have shown that synthetic peptides, and particularly, IL-8 amino terminal peptides, inhibit IL-8 binding to neutrophils and neutrophil chemotaxis (Miller et al., 1990; Miller et al., 1993). An N-terminal pentapeptide IL-8 inhibitor has also been reported (Goodman et al., 1991). Unfortunately, to date, the inhibitory function of IL-8 derived peptides has proven incomplete and insufficient.
As particularly effective peptide inhibitors of CXC intercrines such as IL-8 have yet to be identified, it seems to be clear that compositions other than the IL-8 molecule itself now need to be investigated. The identification of peptide inhibitors capable of preferentially inhibiting neutrophil enzyme release in comparison to chemotaxis would be a particularly advantageous discovery as this would enable neutrophils to enter the lungs and defend against bacterial invasion and yet not cause tissue damage.
The present invention seeks to overcome the drawbacks inherent in the prior art by providing new methods and compositions for modulating and inhibiting the actions of CXC intercrine molecules such as IL-8, GRO (GRO/MGSA) and MIP2xcex2. The peptides and pharmacological compositions disclosed reduce IL-8, GRO and MIP2xcex2 binding to neutrophils and inhibit IL-8-induced neutrophil activation. These peptide formulations are particularly advantageous as they are capable of inhibiting IL-8-induced enzyme release at significantly lower concentrations than is required to inhibit neutrophil chemotaxis. Also provided are methods for treating various diseases and disorders, particularly inflammatory diseases, in which the unrestrained actions of CXC intercrines play a role. Additionally, certain peptides have been found to affect melanoma cell growth. In particular, a novel hexapeptide, Antileukinate (Ac-RRWWCR-NH2), has been found to be a potent inhibitor of binding of xcex1-chemokines to the receptors. The effect of Antileukinate on MGSA/GROxcex1 binding to melanoma cells in several cell lines, including Hs 294T, RMPI-7951, A375P, A375SM, C8161 and WM115. suppresses the growth of the melanoma cells. Additionally, the hexapeptide also inhibits growth in lung adenocarcinoma cell lines A549, NCI-H441 and KS-LU-1 as well as in squamous cell lung cancer NCI-H292 and adenocarcinomas from stomach AGS and Hs746T, breast tissue MCF-7, prostate DU145 and colon Caco-2.
A hexapeptide, termed Antileukinate, has been identified as a potent inhibitor of binding of xcex1-chemokines to their receptors on neutrophils. When Antileukinate was added to melanoma cells, it inhibited the binding of MGSA/GROxcex1. The growth of cells from several melanoma cell lines was suppressed completely in the presence of 100 xcexcM peptide. The cell growth inhibition was reversed by the removal of the peptide from the culture media or by the addition of an excess amount of MGSA/GROxcex1. The viability of Hs 294T cells in the presence of 100 xcexcM peptide was greater than 92%. These findings support the view that MGSA/GROxcex1 is an essential autostimulatory growth factor for melanoma cells. Antileukinate thus appears to inhibit tumor cell growth by preventing binding of the natural ligand to its receptors
The invention is generally based upon the inventors surprising discovery that relatively small peptides including the amino acid sequence Arg Arg Trp Trp Cys Xaa1 (RRWWCX; SEQ ID NO:23), wherein Xaa1 is any amino acid residue, are potent inhibitors of CXC intercrine molecules such as IL-8. As used herein, the terms xe2x80x9cCXC intercrine family moleculesxe2x80x9d and xe2x80x9cCXC intercrinesxe2x80x9d are used collectively to refer to the group of peptide intercrines which include the CXC sequence motif in their N-terminal regions. CXC intercrines are known to include IL-8, GRO, MIP2xcex1, MIP2xcex2 and ENA78, all of which molecules, and any other intercrine polypeptides that include the CXC motif, will be understood to fall within this term as used in the present application.
The inhibitory peptides of the present invention may be termed xe2x80x9cantileukinatesxe2x80x9d. Certain hexamer peptides of the sequence RRWWCX (SEQ ID NO:23) have been previously shown to have anti-bacterial activity against Staphylococcal aureus (Houghten et al., 1991). However, there was no previously documented information to suggest that any such peptides would have the advantageous anti-cytokine/intercrine, anti-neutrophil and anti-inflammatory activities disclosed herein. The anti-tumor activity is particularly unexpected, particularly since the peptides are effective against a wide range of tumor cell types. It is believed that this broad activity arises because the so-called antileukin peptides herein disclosed inhibit binding of several different cytokines that affect cell growth.
In certain aspects, the present invention therefore concerns methods for inhibiting CXC intercrines, such as GRO and MIP2xcex1 or MIP2xcex2, and most particularly, methods for inhibiting IL-8. As used herein, the term xe2x80x9cinhibiting CXC intercrinesxe2x80x9d refers to the processes by which the biological actions of the CXC intercrines are reduced. This may be particularly assessed by inhibiting their binding to one of the IL-8 receptors on their target cells, such as neutrophils, although any mode of determining CXC intercrine inhibition may be employed.
The term IL-8 is used to refer to the cytokine compositions previously known as neutrophil-activating factor, monocyte-derived neutrophil-activating peptide, monocyte-derived neutrophil-chemotactic factor and neutrophil-activating peptide-1. As used herein, the term xe2x80x9cinhibiting IL-8xe2x80x9d generally refers to the processes by which the biological actions of IL-8 are reduced or lessened. This includes the inhibition of any or all of the known actions of IL-8. These actions include modulating sub-cellular effects, such as receptor binding or altering cytosolic calcium levels; modulating cellular effects such as granulocyte recruitment and activation; and also affecting physiological effects, such as inflammation and angiogenesis.
In preferred embodiments, the inhibition of IL-8 function referred to in this application is the inhibition of IL-8 action on granulocytes such as neutrophils (polymorphonuclear neutrophils, PMN). This may be determined in many cellular and physiological ways, as disclosed herein. For example, by measuring inhibition of IL-8 binding to purified receptor compositions or neutrophils; by determining the inhibition of IL-8-induced neutrophil chemotaxis or diapedesis; by measuring the inhibition of IL-8-stimulated neutrophil enzyme release (e.g., myeloperoxidase, xcex2-glucuronidase or elastase release) or superoxide production; or by assaying for anti-inflammatory effects in vivo, e.g., using a rabbit model of dermal inflammation.
The preferred manner of determining IL-8 inhibition, or indeed GRO or MIP2xcex2 inhibition, is to assay for a reduction in the intercrine binding to neutrophils, which is the most simple and straightforward method. In addition, binding of the particular intercrine to its receptor(s) must precede any other action that it has on neutrophils or other cell types. xe2x80x9cInhibitionxe2x80x9d of intercrines, as exemplified by the inhibition of IL-8, GRO or MIP2xcex1 or MIP2xcex2 binding to neutrophils, refers to the capacity of a given peptide or composition to inhibit intercrine binding to any detectable degree, i.e., to reduce binding below the levels observed in the absence of the peptide or composition.
The inhibition of CXC intercrine binding to neutrophils may be expressed as a % Binding Inhibition value, with the higher figures representing the more effective inhibitors. The preferred peptides will generally have the higher % binding inhibition figures. Naturally, the % binding inhibition calculated will depend upon the precise assay conditions, such as the concentration of CXC intercrine and the concentration of the given peptide or composition. Conditions such as those used to generate the data of Table 1A, Table 1B, Table 5A and Table 5B, may be employed to determine whether a given peptide has any inhibitory activity. However, one may choose to employ more discriminatory conditions, such as those using lower peptide concentrations, e.g., on the order of about 20 xcexcM (as used to generate the data of FIG. 1 and FIG. 9), where one desires to obtain particularly accurate quantitative or comparative data. In any event, the determination of whether a peptide or analogue is capable of inhibiting a CXC intercrine, such as IL-8, is a straightforward matter readily achieved using assays such as those disclosed herein.
Although an understanding of the mechanism of action of the CXC intercrine inhibitors is not relevant in terms of their practical utility, it is, however, important to note that the peptide inhibitors of this invention are capable of preferentially inhibiting IL-8-induced neutrophil enzyme release at lower concentrations than IL-8-induced chemotaxis. In this sense, the term xe2x80x9cinhibitingxe2x80x9d, when used in connection with this invention, also means xe2x80x9cmodulatingxe2x80x9d in that certain neutrophil functions are more significantly inhibited than others.
The ability of the peptides to inhibit IL-8-induced neutrophil degradative enzyme release at about a 25 times lower concentration than is required to inhibit IL-8-induced neutrophil chemotaxis is an important discovery that could not have been predicted from prior studies. This means that neutrophils may still be recruited to a site of injury, but that the detrimental effects of the enzymes that they would normally release will be significantly reduced. This property, coupled with their small size, renders these type of peptides ideal for use in various treatment protocols and especially in the treatment of lung injury.
To achieve CXC intercrine inhibition, such as IL-8, GRO or MIP2 inhibition, or to preferentially reduce neutrophil enzyme release in comparison to neutrophil chemotaxis, in accordance with this invention one would generally contact the CXC intercrine family molecule or intercrine target cells, such as granulocytes or neutrophils, with a biologically effective amount of a composition comprising a peptide of the family disclosed herein. The xe2x80x9ccontactxe2x80x9d process is the process by which the active peptide or peptides from within the composition contact either the CXC intercrine peptide or one of their receptors present on a target cell, or both, and reduce or inhibit their functional interaction. Although of scientific interest, the mechanisms by which the CXC intercrine signals transmitted to a given cell are reduced are not relevant to the practice of the invention.
To contact a CXC intercrine or intercrine target cell with a peptide-containing composition one may simply add the peptide or composition to target cells, such as neutrophils, and intercrines in vitro. Alternatively, one may administer a biologically effective amount of a pharmacologically acceptable form of the peptide or composition to an animal, where it will contact, e.g., neutrophils or macrophages and intercrines in a biological fluid in vivo. In this context, xe2x80x9ccontactxe2x80x9d is achieved simply by administering the composition to the animal. Virtually any pharmaceutical peptide formulation may be used, including, but not limited to, formulations for parenteral administration, such as for intravenous, intramuscular and subcutaneous administration; inhalants, aerosols and spray formulations; formulations of peptides for topical use, such as in creams, ointments and gels; and other formulations such as peptides with lipid tails, peptides encapsulated in micelles or liposomes and drug release capsules including the active peptides incorporated within a biocompatible coating designed for slow-release.
Increased levels of IL-8 are known to be present in lung edema fluids in patients with ARDS (Miller et al., 1992) and in the sputum of patients with cystic fibrosis (Richman-Eisenstat et al., 1993); in pleural spaces of patients with pleural effusions (Miller and Idell, 1993); in joint fluids from patients with several kinds of joint disease (Brennan et al., 1990; Miller and Brelsford, 1993), in psoriatic plaques and in synovial fluid from arthritic patients (Lam et al., 1990). Inappropriate neutrophil activation is connected with all such disorders and with ischemic and reperfusion injuries (DeForge et al., 1992). As the inhibition of IL-8 neutrophil recruitment has been shown to reduce lung inflammation in vivo (Mulligan et al., 1993), and as the type of in vitro studies employed herein are accepted as being predictive of in vivo activity (see U.S. Pat. No. 5,079,228, incorporated herein by reference), the highly successful inhibition of IL-8-induced neutrophil activation disclosed in the application supports the broad clinical utility of these peptides.
The present invention therefore also provides methods for treating a wide variety of diseases and disorders in which CXC intercrines, particularly IL-8, play a role, especially those which have an inflammatory component. This includes treating subjects with lung injuries and disorders, including bronchial inflammation, such as chronic bronchitis, cystic fibrosis, pleural effusions, asthma, and ARDS; skin disorders such as psoriasis and dermatitis; diseases of the joints, including rheumatoid arthritis; and generally reducing inflammation in other clinical settings, such as in the treatment of pseudogout, inflammatory bowel disease or reperfusion cardiac damage after myocardial infarction. These peptides could even be used as anti-proliferative agents to downregulate lymphocyte proliferation, for example, in the treatment of cancer and other diseases and disorders associated with increased cellular proliferation.
To treat any one of the above conditions, or any other disorder influenced by neutrophil activity and characterized by inflammation, one would identify a patient having the particular inflammatory or IL-8-linked disease and then administer to the patient, preferably parenterally, a biologically effective amount of a pharmaceutical composition which includes one or more peptides of the family disclosed herein.
Naturally, one would generally tailor the particular pharmaceutical formulation according to the disease or disorder being treated. For example, in methods to treat skin disorders, a topical cream or gel formulation would be used, whereas in methods to treat pulmonary disorders, injectable formulations, or even a spray, aerosol or inhalant, may be employed. In methods to reduce inflammation in other areas of the body, one may use peptides formulated for parenteral administration or peptides incorporated in a biocompatible coating designed for slow-release. Liposome-encapsulation may be employed, which is known to increase the efficacy and significantly prolong the half-life of administered compounds, particularly those of lower molecular weight such as the peptides disclosed herein. Various compositions and techniques for preparing all such pharmaceutical formulations will generally be known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and associated administrative techniques one may wish to refer to Remington""s Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., incorporated herein by reference.
IL-8 or CXC intercrine inhibition is achieved by using a biologically effective amount of the inhibitory peptide or peptides. As used herein, a xe2x80x9cbiologically effective amountxe2x80x9d of a peptide or composition refers to an amount effective to inhibit the actions of IL-8 or the particular intercrine. For example, in regard to IL-8 inhibition, an appropriate amount would be that effective to reduce neutrophil enzyme release, particularly in comparison to chemotaxis. As disclosed herein, a variety of different peptide concentrations are very effective in vitro, such as those between about 100 xcexcM and about 20 xcexcM. Clinical doses which result in similar a local concentration of peptides are therefore contemplated to be particularly useful.
Naturally, in a clinical context, the quantity and volume of the peptide composition administered will depend on the host animal and condition to be treated and the route of administration. The precise amounts of active peptide required to be administered will depend on the judgment of the practitioner and may be peculiar to each individual. However, in light of the data presented herein, the determination of a suitable dosage range for use in humans will be straightforward. For example, in treating ARDS or cystic fibrosis, doses in the order of about 0.83 mg/kg body weight/hour (mg/kg/hr) to about 16.56 mg/kg/hr, preferably about 0.83 mg/kg/hr to about 4.14 mg/kg/hr, and more preferably about 1.66 mg/kg/hr of active ingredient peptide per individual are contemplated.
The compositions for use in inhibiting CXC intercrines, such as IL-8, GRO and MIP2xcex1 or MIPxcex2, in accordance with the present invention will be compositions that contain a relatively small peptide, generally of from 6 to about 14 residues in length, which includes within its sequence the amino acid sequence RRWWCX (SEQ ID NO:23). The term xe2x80x9ca peptidexe2x80x9d in this sense means at least one peptide, and may refer to one or more such peptides which include a sequence in compliance with the general formula RRWWCX (SEQ ID NO:23).
The relatively small peptides encompassed by the present invention may be any length between six residues and about 14 or 15 or so residues in length, with the precise length not being an important feature of the invention. There are many advantages to using smaller peptides, for example, the cost and relative ease of large scale synthesis, and their improved pharmacological properties, such as the ease with which they can penetrate tissues and their low immunogenicity;
In addition to including an amino sequence in accordance with the sequence RRWWCX (SEQ ID NO:23), the peptides may include other short peptidyl sequences of various amino acids. For example, in certain embodiments, the peptides may include a repeat of the sequence RRWWCX (SEQ ID NO:23) or RRWWCXX (SEQ ID NO:57). They may also contain additional sequences including, e.g., short targeting sequences, tags, labeled residues, amino acids contemplated to increase the half life or stability of the peptide, or indeed, any additional residue desired for any purpose, so long as they still function to inhibit intercrines such as IL-8xe2x80x94which can be readily determined by a simple assay such as those described herein.
Amino acids which may incorporated into the peptides include all of the commonly occurring amino acids. Two designations for amino acids are used interchangeably throughout this application, as is common practice in the art: Alanine=Ala (A); Arginine=Arg (R); Aspartic Acid=Asp (D); Asparagine=Asn (N); Cysteine=Cys (C); Glutamic Acid=Glu (E); Glutamine=Gln (Q); Glycine=Gly (G); Histidine=His (H); Isoleucine=Ile (I); Leucine=Leu (L); Lysine=Lys (K); Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser (S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr (Y); Valine=Val (V).
Any of the so-called rare or modified amino acids may also be incorporated into a peptide of the invention, including the following: 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine (beta-Aminopropionic acid), 2-Aminobutyric acid, 4-Aminobutyric acid (piperidinic acid), 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 2,2xe2x80x2-Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isodesmosine, allo-Isoleucine, N-Methylglycine (sarcosine), N-Methylisoleucine, N-Methylvaline, Norvaline, Norleucine and Ornithine.
The inhibitory compositions of the invention may include a peptide modified to render it biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Pat. No. 5,028,592 (incorporated herein by reference), protected peptides often exhibit increased pharmacological activity, as was found to be true in the present case.
The present invention therefore encompasses compositions comprising an acylated peptide or peptides, and preferably, a peptide acylated at the N-terminus. Although virtually any acyl group may be employed in this context, the inventors have found that the addition of an acetyl group to the N-terminus of a given peptide also renders the resultant peptide surprisingly effective at inhibiting intercrines such as IL-8. The inhibitory peptide compositions may also include a peptide(s) which is amidated at the C-terminus, i.e., to which an NH2 group has been added. In particularly preferred embodiments, peptides which have both an acylated N-terminal and an amidated C-terminal residue are preferred as they are believed to most closely mimic natural protein and peptide structure.
Compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids or a mixture thereof. The finding that peptides composed entirely of D-amino acids have potent inhibitory activity is particularly important as such peptides are known to be resistant to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives. The anti-intercrine and anti-IL-8 compositions of the present invention will generally comprise one or more peptides which include an amino acid sequence in accordance with those set forth in SEQ ID NO:1 or SEQ ID NOs:24 through 42. In certain embodiments, short hexamer peptides may be preferred. In such cases, the inhibitory compositions will generally comprise one or more peptides which have an amino acid sequence in accordance with those set forth in SEQ ID NO:1 or SEQ ID NOs:24 through 42, presented below:
Arg Arg Trp Trp Cys Arg (SEQ ID NO:1)
Arg Arg Trp Trp Cys Ala (SEQ ID NO:24)
Arg Arg Trp Trp Cys Cys (SEQ ID NO:25)
Arg Arg Trp Trp Cys Asp (SEQ ID NO:26)
Arg Arg Trp Trp Cys Glu (SEQ ID NO:27)
Arg Arg Trp Trp Cys Phe (SEQ ID NO:28)
Arg Arg Trp Trp Cys Gly (SEQ ID NO:29)
Arg Arg Trp Trp Cys His (SEQ ID NO:30)
Arg Arg Trp Trp Cys Ile (SEQ ID NO:31)
Arg Arg Trp Trp Cys Lys (SEQ ID NO:32)
Arg Arg Trp Trp Cys Leu (SEQ ID NO:33)
Arg Arg Trp Trp Cys Met (SEQ ID NO:34)
Arg Arg Trp Trp Cys Asn (SEQ ID NO:35)
Arg Arg Trp Trp Cys Pro (SEQ ID NO:36)
Arg Arg Trp Trp Cys Gln (SEQ ID NO:37)
Arg Arg Trp Trp Cys Ser (SEQ ID NO:38)
Arg Arg Trp Trp Cys Thr (SEQ ID NO:39)
Arg Arg Trp Trp Cys Val (SEQ ID NO:40)
Arg Arg Trp Trp Cys Trp (SEQ ID NO:41)
Arg Arg Trp Trp Cys Tyr (SEQ ID NO:42)
In other embodiments, the inhibitory compositions of the invention may include one or more peptides which include a sequence in accordance with the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa2 (SEQ ID NO:2). In these cases one of the variable positions has been defined as arginine and the remaining Xaa2 may be any amino acid residue. Such sequences are exemplified by those set forth in SEQ ID NOs:3 through 22. Where short heptamer peptides are preferred, the compositions will generally comprise one or more peptides which have an amino acid sequence in accordance with those set forth below:
Arg Arg Trp Trp Cys Arg Ala (SEQ ID NO:3)
Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO:4)
Arg Arg Trp Trp Cys Arg Asp (SEQ ID NO:5)
Arg Arg Trp Trp Cys Arg Glu (SEQ ID NO:6)
Arg Arg Trp Trp Cys Arg Phe (SEQ ID NO:7)
Arg Arg Trp Trp Cys Arg Gly (SEQ ID NO:8)
Arg Arg Trp Trp Cys Arg His (SEQ ID NO:9)
Arg Arg Trp Trp Cys Arg Ile (SEQ ID NO:10)
Arg Arg Trp Trp Cys Arg Lys (SEQ ID NO:11)
Arg Arg Trp Trp Cys Arg Leu (SEQ ID NO:12)
Arg Arg Trp Trp Cys Arg Met (SEQ ID NO:13)
Arg Arg Trp Trp Cys Arg Asn (SEQ ID NO:14)
Arg Arg Trp Trp Cys Arg Pro (SEQ ID NO:15)
Arg Arg Trp Trp Cys Arg Gln (SEQ ID NO:16)
Arg Arg Trp Trp Cys Arg Arg (SEQ ID NO:17)
Arg Arg Trp Trp Cys Arg Ser (SEQ ID NO:18)
Arg Arg Trp Trp Cys Arg Thr (SEQ ID NO:19)
Arg Arg Trp Trp Cys Arg Val (SEQ ID NO:20)
Arg Arg Trp Trp Cys Arg Trp (SEQ ID NO:21)
Arg Arg Trp Trp Cys Arg Tyr (SEQ ID NO:22)
The invention also contemplates the use of peptides having the amino acid sequence Gln Ile Pro Arg Arg Ser Trp Cys Arg Phe Leu Phe (SEQ ID NO:52), either alone, or more preferably, in combination with one or more of the other peptides described above. The successful use of this dodecamer illustrates both the fact that longer peptides are successful and that certain biologically functional equivalent peptides are active. All such active equivalents therefore fall under the scope of the present invention.
The compositions for use in the inhibitory methods described herein may contain only a single active peptidyl species. Alternatively, they may contain more than one peptide, up to and including about 40 or 45 or so distinct peptides. Any and all of the various combinations are contemplated, such as compositions comprising 2, 3, 5, 10, 15, 20, 30 or 45 or so distinct peptides.
Compositions comprising peptides having the amino acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID NO:1) and/or the amino acid sequence Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO:4) are contemplated to be particularly useful, although the invention is not limited to these peptides in any way. In this regard, it is important to note that considerations other than in vitro activity, such as plasma half life and stability, may be considered in ultimately choosing peptides which are preferred for clinical embodiments. The effects of different amino acid substitutions on these parameters may be readily determined and the results used to design the optimum peptide or combination of peptides for use in vivo.
The RRWWCX (SEQ ID NO:23) sequence element is an important feature of the peptides of this invention. However, this does not exclude certain biological functional equivalents from falling within the scope of the invention. For example, the inventors have discovered that the first tryptophan in RRWWCX (SEQ ID NO:23) can be exchanged, e.g., by replacing with serine, with only modest loss of activation. Therefore, one example of equivalents encompassed by the invention are peptides of the sequence RRXWCX (SEQ ID NO:58). xe2x80x9cEquivalent amino acidsxe2x80x9d may be defined as amino acids whose hydrophilic or hydropathic index are within xc2x12, more preferably, within xc2x11, and most preferably, within xc2x10.5 of each other. Of course, to be a xe2x80x9cfunctional equivalentxe2x80x9d, a peptide must still retain its intercrine or IL-8 inhibitory activity, as may be easily determined using assays such as those disclosed herein.
In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds, called peptidomimetics, may be formulated to mimic the key portions of the peptide structure. Such compounds may be used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
The peptides and compositions for use in the invention may be prepared by any one of a variety of different methods. One preferred method for preparing peptides in accordance with the present invention is contemplated to be via automated peptide synthesis. A synthetic peptide may be straightforwardly prepared using an automated peptide synthesizer, the operation of which will be generally known to those of skill in the art. This method is one of those generally preferred for preparing large quantities of a given peptide, e.g., once a particular peptide has been chosen for therapeutic use.
Another preferred method for preparing inhibitory peptides, and the biological functional equivalents thereof, is to use a combinatorial peptide library method, as described by Houghten et al. (1991) and disclosed in International Patent Application PCT WO 92/09300, the entire disclosure of which is specifically incorporated herein by reference. These methods are particularly useful for preparing and analyzing a plurality of peptides having a substantially predetermined sequence, such as RRWWC, to which is appended a variety of different amino acids at one or more positions. These methods may be used to synthesize a peptide mixture for direct use in the formulation of a composition in accordance with the invention or to identify a particularly active peptide for subsequent individual synthesis.
If desired, peptides may also by prepared by molecular biological means and the xe2x80x9crecombinantxe2x80x9d peptide obtained from recombinant host cells which express the peptide. To achieve this, one would prepare a specific oligonucleotide, based upon the sequence of the desired peptide, as is known to those of skill in the art, and then insert the oligonucleotide into an expression vector, such as any one of the many expression vectors currently available commercially. One would then transform a prokaryotic or eukaryotic host cell with the vector, where it will direct the expression of the so-called recombinant version of the peptide, which may then be purified from the recombinant host cell. This methodology is standard practice in the art (see e.g., Sambrook et al., 1989).